Program Summary There is still much unknown about how nitric oxide (NO) production by nitric oxide synthase (NOS) is tightly regulated. This is remarkable because unregulated NO production by NOS in vivo is a critical problem in an increasing number of diseases lacking effective treatments, including cancer and cardiovascular diseases. Before logically designing effective preventive and therapeutic strategies targeting unregulated NO production, one must clearly understand the control mechanisms of NOS catalysis. An important component of the function of the NOS enzyme is the regulation of interdomain electron transfer (IET) processes required for NO synthesis. The long-term goal of this project is to investigate the mechanisms of the crucial IET processes in NOS at the molecular level, in order to determine the key sequences for controlling the NOS function. It is proposed that the calmodulin (CaM) activation of NO synthesis in endothelial and neuronal NOS (eNOS and nNOS) requires a conformational change of the flavin mononucleotide (FMN) domain from its original electron- accepting (input) state to a new electron-donating (output) state. The putative output state is envisioned as a complex between the FMN binding and oxygenase domains, thus facilitating efficient IET between the FMN and the catalytic heme in the oxygenase domain. The FMN-heme IET within the NOS output state is essential for NO synthesis. However, the mechanism of the output state formation remains unclear, which thus constitutes a critical barrier for understanding the CaM controlled NOS catalytic mechanisms more completely. The focus of this study is to investigate the mechanisms of CaM-activated output state formation at the molecular level. We hypothesize that specific CaM binding and productive FMN/heme interactions are two critical structural determinants for formation of the NOS output state. This hypothesis will be tested by quantitating the FMN-heme IET kinetics in a well-validated model of the NOS output state through two complementary and synergistic Aims. We have developed innovative laser flash photolysis approaches to directly determine the FMN-heme IET within the NOS output state. The experimental design will integrate our laser flash photolysis approach and biophysical techniques with site-directed mutagenesis, in order to determine mechanistic roles of specific amino acids in CaM-controlled formation of the output state in eNOS and nNOS. The proposed studies will significantly improve the fundamental understanding of NOS regulation, and will provide important new insight as to how NOS might be selectively modulated for therapeutic purposes.